摘要
目的 构建携带人酪氨酸羟化酶 (tyrosinehydroxylase, TH)目的基因片段的真核表达重组质粒—pcDNA3. 1his hTH,与pEGFP- C2共转染恒河猴骨髓源神经干细胞,观察hTH基因在骨髓源神经干细胞中的表达情况。方法 应用基因重组技术,将pWAV2-TH中TH基因亚克隆到pcDNA3 .1his真核表达载体,以酶切和测序方法鉴定重组质粒pcDNA3 .1his- hTH的正确性;将pcDNA3. 1his hTH和pEGFP- C2经电击穿孔法共转染恒河猴骨髓源神经干细胞, 24h后观察EGFP瞬时表达情况, 10d后进行hTH基因RT PCR,以及hTH和 6His单克隆抗体的免疫组化检测。结果⑴酶切和测序结果均证实pcDNA3 .1his hTH的正确性;⑵细胞转染 24h后,荧光显微镜下可观察到EGFP的表达, 80%以上细胞发出绿色荧光; 10d后RT PCR检测到细胞内有hTH基因的表达,免疫组化结果显示细胞有hTH和 6His抗原表达。结论 成功构建的pcDNA3 .1his -hTH和pEGFP- C2能够共转染恒河猴骨髓源神经干细胞,hTH、EGFP和 6His基因在细胞内有效表达。该系统可以作为体外检测转染率、细胞移植治疗帕金森病活体跟踪移植细胞的技术平台。
Objective To construct a recombinant plasmid pcDNA3.1his-hTH which can express hTH (human tyrosine hydroxylase) gene and 6-his in eukaryocyte and cotransfect neural stem cells derived from bone marrow stromal cells (NSCs-BMSCs) with pEGFP-C2, then observe the expression of hTH gene in NSCs-BMSCs. Methods With the technology of gene re-arrangement, hTH gene in pWAV2-TH plasmid was subcloned into pcDNA3.1his vector to obtain pcDNA3.1his-hTH plasmid, then its correctness was evaluated by the means of restriction enzyme analysis and sequencing. pcDNA3.1his-hTH and pEGFP-C2 was contransfected into NSCs-BMSCs by NucleofectorTM. After 24h, the transient expression of EGFP was observed under fluorescence microscope,and the antigen expression of TH and 6-his were measured by immunohistochemistry.Meanwhile, the mRNA of TH gene was detected by RT-PCR. Results ⑴ Correct construction of pcDNA3.1his-hTH was identified by methods of restriction enzyme analysis and nucleotide sequence determination.⑵ Green fluorescence was emitted from transfection cell under fluorescent microscope after 24h, then immunohistochemistry showed large parts of transfection cells which expressed TH and 6-his and displayed positive staining, and the expression of hTH gene was detected by RT-PCR after 10d . Conclusions pcDNA3.1his-hTH, which was successfully constructed, and pEGFP-C2 were cotransfected in NSCs-BM of Rhesus monkey. The system could be as a technic platform to detect transfection efficiency in vitro and track grafted cells to treat PD in vivo, because hTH、EGFP and 6His genes were able to be expressed in NSCs-BMSCs.
出处
《中华神经外科杂志》
CSCD
北大核心
2005年第2期118-122,共5页
Chinese Journal of Neurosurgery
基金
广东省科技厅团队基金(200025)
国家自然科学基金(30270491)
关键词
PCDNA3
共转染
EGFP
神经干细胞
TH基因
骨髓
表达
构建
结论
情况
Human tyrosine hydroxylase gene
Neural stem cells derived from bone marrow (NSCs-BM)
Nucleofector TM technology
Cotransfection
Rhesus monkey
